Tissue engineering may be defined as the art of reconstructing mammalian tissues, both structurally and functionally (Hunziker, 2002). Tissue engineering includes the provision of cells or of a natural or synthetic scaffold that serves as an architectural support onto which cells may attach, proliferate, and synthesize new tissue to replace tissue losses due to disease, trauma or age.
The trend in tissue engineering in general is to utilize biomaterials to promote controlled healing or tissue regeneration. In orthopedics and dentistry the clinical focus transforms from traditional metal and other inorganic implants, plates, screws and cements to biologically based products for mineralized tissue regeneration.
Natural polymers are of major interest in tissue engineering since they tend to be biocompatible and biodegradable and may have the potential to enhance cell adhesion and proliferation. Additionally, such material substrates can be prepared in various forms and shapes, including strips, sheets, sponges and beads for implantation.
Bone
Bone is a unique type of tissue that comprises both organic and inorganic phases, that undergoes modeling and remodeling wherein old bone is lost (resorption) and new bone is formed (formation/replacement). Bone formation may be enhanced either by recruiting osteoblasts, the bone forming cells, or by inhibiting recruitment or activity of osteoclasts, the bone resorbing cells. Osteoblasts and osteoclasts work together in a coordinated fashion to form and remodel bone tissue. Bone repair or replacement is a viable consideration in indications including osteopenia, osteoporosis, bone tumors, spinal fusion and fractures.
Biomineralization of Bone
Biomineralization refers to the deposition of inorganic solids in biological systems. Mann (2001) has defined the term biologically controlled mineralization as the highly regulated process that produces materials such as bones, shells and teeth that have specific biological functions and structures. Biologically controlled mineralization is characterized by specific crystalline and chemical properties, which may include: rather uniform particles size, well-defined structures and compositions, high level of spatial organization, preferred crystallographic orientation and higher order assembly into hierarchical structures.
Hydroxyapatite (HA), having the chemical formula Ca10(PO4)6(OH)2, is one of the major constituents of the inorganic components in bone, as well as in other human hard tissues (Posner, 1969; Mann, 2001). HA enables formation of bone on its surface by supporting attachment, migration, proliferation, and differentiation of bone forming cells (Oliveira, 2003; Delange, 1989). The mechanical properties of HA in absence of the organic matrix onto which it deposits in vivo, does not resemble natural human bone. HA is rigid and often very brittle and thus cannot be used per se for weight-bearing applications (Oliveira, 2003).
The natural mineralization of bone is considered to occur by deposition of HA or its precursor forms in an organic extracellular matrix composed of collagen and other proteins, many of which are rich in acidic residues (Hunter, 1996; Teraub, 1989). The major role of collagen is to render the bone improved mechanical properties through an hierarchical composition of the organic fibers and aligned HA minerals (Lowenstam et al., 1989, Mann, S., 2001, Teraub 1989). Non-collagenous proteins isolated from bone extracellular matrices that are rich in acidic amino acids i.e. bone sialoprotein, osteopontin, osteocalcin, osteonectin and others (Young et al, 1992), have been proposed to be involved in the nucleation, and growth of carbonated apatite (Hunter et al., 1996). Among these, sialoprotein (BSP), a glycosylated and sulphated phosphoprotein, found almost exclusively in mineralized connective tissues, is the most widely accepted protein linked to apatite nucleation (Ganss et al., 1999). BSP exhibits fragments rich in both glutamic- and aspartic-acid residues (Oldberg et al, 1988) as well as the cell binding arginine-glycine-aspartate (RGD) motifs. Despite numerous studies aiming at unraveling the principles of apatite biomineralization, detailed mechanisms that account for the role of acid rich proteins in this process, are yet to be elucidated.
Among the main properties of organic interfaces that may be contributing to nucleation of biominerals are electrostatic accumulation and structural correspondence. Electrostatic accumulation is considered to be the initial step in biomineralization. It is believed that one of the most essential properties of bone acid-rich proteins and possibly also collagen is their ability to control nucleation by charged amino acid residues on their surfaces. The primary residues are acidic and phosphorylated amino acids, which at biological pH, may expose charged functional groups, i.e. negatively charged carboxylate groups of glutamic acid and aspartic acid as well as negatively charged phosphates. (Addadi, 1985; Mann, 1988).
Many materials have been utilized for bone repair. Synthetic materials are being developed in order to replace autologous harvesting problems and the health risks attendant with allogeneic material. Inorganic materials such as calcium phosphate and hydroxyapatite have been utilized as bone and dental fillers (reviewed in LeGeros, 2002) but lacking many of the extra cellular like functionalities, none can be considered entirely satisfactory in meeting the criteria required for successful tissue engineering.
Recent developments in the study of peptide self-assembly matrices have advanced the understanding of the relationship between amino acid composition, molecular assembly forms and interaction of these materials with cells. Certain peptides and proteins have been shown to promote osteogenic cell adhesion. A 15-mer peptide fragment of collagen 1α1 has been designed to include cell binding domain for mesenchymal progenitor cells. This fragment is commercially available as Pepgen P-15® in combination with an organic bovine derived bone matrix (ABM) as particles or cement for bone grafting in patients with periodontal osseous defects (Valentin and Weber, 2004).
Gilbert et al. (2000) teach a fusion peptide of two extracellular matrix proteins, statherin and osteopontin that binds hydroxyapatite and mediates cell adhesion. The chimeric peptide was shown to have utility in tissue engineering and vaccine applications.
Goldberg et al. (2001) teach synthetic poly-L-glutamic acid and poly-L-aspartic acid peptides and their ability to bind hydroxyapatite. He et al. (2003) report that the acidic protein dentin matrix protein 1 (DMP 1) assembles into acidic clusters that are claimed to nucleate the formation of hydroxyapatite in vitro.
International patent application WO 2005/003292 relates to a composition useful for making homogenously mineralized self-assembled peptide amphiphile nanofibers and nanofiber gels. The peptide amphiphiles comprise three regions: an alkyl tail at the N-terminus providing the peptide with a hydrophobic nature, a tetra cysteine region, and a C-terminal sequence which includes cell adhesion or crystal nucleation sequences. Due to their amphiphilic nature the peptides self assemble into nanofiber matrices which may be prepared with appropriate phosphate and calcium solutions to yield mineral templated matrices.
U.S. Pat. Nos. 5,670,483; 5,955,343; 6,548,630 and 6,800,481 relate to amphiphilic peptides having alternating hydrophobic and hydrophilic residues, and their resultant macroscopic membranes, respectively. Specifically, two peptides having the amino acid sequences (AEAEAKAK)2 and (ARARADAD)2 were shown to self assemble into macroscopic membranes useful for in vitro culturing of cells and biomaterial applications. The former sequence was originally found in a region of alternating hydrophobic and hydrophilic residues in a yeast protein called zuotin.
US Patent Publication No. US 2005/0181973 discloses a self-assembling peptide comprising two domains, the first one comprising complementary alternating hydrophobic and hydrophilic amino acids that are overall are neutrally charged with equal number of positively and negatively charged amino acids, that self-assemble into a macroscopic structure, including hydrogels, when present in unmodified form; and a second amino acid domain comprising a biologically active peptide motif or a target site for an interaction with a biomolecule. That application further teaches that replacement of the positively charged residues, lysine (K) and arginine (R), by negatively charged residues, such as aspartate (D) and glutamate (E), prevents peptide self-assembly into macroscopic structures and only β-sheet and not macroscopic structures are formed in the presence of salt. The VE20 peptide, a 20-mer peptide comprising alternating valine (V) and glutamate (E) amino acids, was disclosed as not able to self-assemble to form macroscopic structures.
US Patent Publication No. US 2006/0025524 discloses a method for making a hydrogel from a solution of peptides, mainly peptides containing Val-Lys repeats or peptides with at least one positively-charged residue per 6 amino acids, which undergo change in conformation from random coil to β-hairpin secondary structures, that promote hydrogel formation. The hydrogel is formed by alteration peptide concentration or one or more environmental signals or stimuli (e.g., change in pH, ionic strength, specific ion concentration, and/or temperature of the solution,).
The “RGD” (Arg-Gly-Asp) tri-peptide sequence, which occurs in fibronectin and has been shown to promote cell adhesion and growth, has been disclosed in inter alia, U.S. Pat. Nos. 4,988,621; 4,792,525 and 5,695,997. U.S. Pat. No. 6,291,428 teaches peptides comprising the RGD amino acid sequence for promoting in situ bone mineralization.
The inventor of the present invention reported amphiphilic peptides that form β-strand monolayers when spread at air-water interfaces (Rapaport, 2000; Rapaport, 2002). Peptides of seven to 17 amino acid residues were found to form crystalline arrays with coherence lengths of about 100 to about 1000 Å. A 30-residue peptide, which incorporates proline residues to induce reverse turns, was designed to form an ordered triple stranded β-sheet monolayer at the air water interface. The formation of hydrogels from these peptides was neither taught nor suggested in those publications.
There is an unmet medical need for multifunctional biomaterials which may be fabricated in various clinically relevant forms such as hydrogels, membranes or solid matrices and mineral-peptide composites, useful for promoting both osteogenic cell activity and biomineralization, in situ.